Nghiên cứu thực trạng, một số yếu tố liên quan đến nhiễm giun lươn strongyloides spp và kết quả điều trị bằng ivermectin tại huyện đức hoà, tỉnh long an (2017 – 2018) tt tếng

1 INTRODUCTION Strongyloides spp is a chronic causative infectious agent This agent is considered to be one of the neglected tropicals infectious pathogens, but it is an important public health problem due to the characteristic of the autoimmune cycle, leading to hyper infection and death in some patients People are acquired Strongyloides spp agent through contact with contaminated soil sources directly such as agricultural cultivation, recreation activities, etc The adult worms usually located in the intestine wall, causing abdominal pain, prolonged diarrhea, or colitis, In addition to disease at the gastrointestinal tract, the stage when infective larvae of Strongyloides spp enter the human body, can move to many organs, causing very diverse clinical diseases The problem of accurate diagnosis of the case therefore also faces many difficulties Treatment strongyloidiasis cases is more difficult than other intestinal helminths, especially with hyper infection The treatment of cases has still been inconsistent with the timing of treatment and drug selection Although strongyloidiasis was discovered in Southern Vietnam at first time, but there has not been many studies on this pathogen recently According to the study results in Cu Chi district, HCM city previously, the prevalence of Strongyloides spp infection in the community was quite high While Duc Hoa district of Long An province was located bordering with Cu Chi district, HCM city, there still has not been any research on these pathogens Stemming from this reality, we conducted the thesis: Studying the actual situation, various factors related to human Strongyloides spp infection and ivermectin effectiveness in Duc Hoa district, Long An province (2017 – 2018).”with the objective: Determine the actual situation and factors related to human Strongyloides spp infection in Duc Hoa district, Long An province in 2017-2018 Determine component species of Strongyloides in human strongyloidiasis disease by morphological characterization and molecular biology Describe the clinical symptoms, para-clinical and evaluate results of treatment for strongyloidiasis by ivermectin single dose 2 NOVELTY, SCIENTIFIC AND PRACTICAL SIGNIFICANCE OF THE THESIS The thesis provides details of valuable scientific data on the status of Strongyloides spp infection and related factors in many research sites of Duc Hoa district, Long An province Provide details of the presence of symptoms, signs and value of para-clinical test in cases of gastrointestinal strongyloidiasis Determine the clinical efficacy of ivermectin and the effectiveness of larval clearance, which is the basis for developing an appropriate intervention program For the first time, a new species of Strongyloides spp was identified from patient in Duc Hoa district that derived from animal origin, by using molecular biology techniques THESIS STRUCTURE The thesis consists of 136 pages divided into the following sections: Introduction (02 pages), Chapter 1: Literature review (31 pages), Chapter 2: Study subjects and methods (29 pages), Chapter 3: Study results (34 pages), Chapter 4: Discussions (36 pages), Conclusions (02 pages), and Recommendations (1 page) There are 36 tables, 13 figures, and 115 references (12 pages, including 28 Vietnamese documents and 87 English documents) Chapter LITERATURE REVIEW 1.1 History of discovering strongyloidiasis In July 1876, Louis Normand found the parasite in the stool samples of French diarrhea patients with a history of coming to the Southern Vietnam He named this agent Anguillula stercoralis and the corresponding disease has been called Cochin -China diarrhea Coming to 1915, the council named scientific unification named this pathogen Strongyloides stercoralis 1.2 Pathogen There are about 104 species of Strongyloides spp, including 52 common species, some of which cause disease in domestic pets and other animals The main pathogen cause human disease is S stercoralis, and less is S fuelleborni Some other species such as S procyonic (host is raccoons), S myopotami and S ratti (hosts are rats and rodents) are considered to be parasitic diseases transmitted from animals to humans 1.2.1 Morphology The development stages of Strongyloides spp including: parasitic female living worm, free-living female worm, free-living male worm, the first stage larvae (rhabditiform), the second stage larvae (filariform) and eggs Parasitic male worm has still been not found 1.2.2 Biological development cycle of Strongyloides spp Strongyloides spp have two stages of the pathogenesis cycle: the parasitic cycle and the free living cycle Free living cycle often happen in the tropics due to adaptive with conditions of external environment In addition, strongyloidiasis also has autoinfection cycle The autoinfection cycle occurs when all or some of rhabditiform larvae reside in the intestinal wall, rapidly molt to the infected stage, establishing a parasitic development stage inside the host and this phenomenon may remain maintenance of host life This phenomenon also often occurs in patients who have immunosuppression status This autoinfection process leads to two severe status of strongyloidiasis: hyperinfection syndrome and disseminated strongyloidiasis 1.3 Epidemiological characteristics The infection prevalence is usually less than 1% in temperate zone, but may be above 25% in many parts of the tropics 1.3.1 The situation of Strongyloides spp infection in the world Strongyloidiasis is an infectious disease in many countries, especially in West Africa, the Caribbean, Southeast Asia, tropical regions of Brazil, Cambodia and Spain Southeast Asia area has the highest prevalence of the disease 1.3.2 The situation of Strongyloides spp infection in Vietnam According to a Galliard survey in 1940, in the northern Vietnam, the prevalence of Strongyloides spp infection was from 0.2 to 2.5% of the population Recent studies using ELISA technique have shown that the infection rate is higher than 7.6-10.9% Strongyloides spp are widely distributed in the southern provinces of Vietnam, provinces such as Long An, Binh Duong, Tay Ninh, Dong Nai and Ho Chi Minh City (Cu Chi, Thu Duc and Hoc Mon districts) and some provinces in the Central region and Highlands 1.4 Strongyloidiasis pathology The strongyloidiasis has an incubation period approximately month The majority of cases in endemic areas often have no symptoms or vague symptoms Strongyloidiasis is divided into two forms: 1.4.1 Chronic, uncomplicated Strongyloidiasis: happen in normal individuals, without immunodeficiency, possible symptoms include: Skin manifestations: The cutaneous larvae migrans, skin bruises, nonspecific urticaria, Gastrointestinal manifestations: Abdominal pain, diarrhea, weight loss, anal itching Other manifestations: Patient coughing, pneumonia, signs of cachexia, 1.4.2 Severe disease, complications This form of the disease is common in immunocompromised individuals who use corticosteroids, immunosuppressing or accompanied by other chronic diseases such as chronic obstructive pulmonary disease, chronic kidney failure, malignancy, diabetes, alcoholism, malnutrition 4 1.4.3 Hyperinfection syndrome and disseminated strongyloidiasis Hyperinfection syndrome is manifestted by a phenomenon in which the rapid increasing the number of pathogens leads to an excessive burden without the spread of external larvae Disseminated strongyloidiasis is the ultimate consequence of hyperinfection syndrome Affected organs include the lungs, liver, heart, kidneys, endocrine organs and central nervous system Strongyloidiasis is severe and lead to death 1.5 Paraclinical diagnosis Diagnostic indicators can be based on nonspecific tests: increased number of eosinophils, increased CRP, increased serum IgE, etc 1.5.1 Direct test Strongyloides spp larvae are usually found in faeces or in gastric, duodenum, sputum, biopsy tissue at a gastric ulcer or other tissue, etc Direct tests include as follows: direct stool test, Baermann concentration technique, Formalin - ether, Harada - Mori stool culture or agar plate culture 1.5.2 Indirect diagnosis Currently, serological testing methods are popular and widely used due to their high sensitivity and general applicability The most widely used technique is the ELISA test to detect immunoglobulin G (IgG) antibodies 1.5.3 Molecular biology diagnosis The PCR test detects parasitic DNA in feces, especially Strongyloides spp, which has the advantage of high sensitivity and specificity PCR techniques have been used include as follows: real-time PCR, nested PCR, multiplex real-time PCR and multiplex PCR 1.6 Treatment Treatment human strongyloidiasis was based on using current drugs: Ivermectin 150-200 µg/kg/ day single dose, albendazole 10-15 mg/kg/day and thiabendazole 50 mg/kg/day The dose of each drug is determined but no consistent guide about the number of treatment days CHAPTER STUDY SUBJECTS AND METHODS 2.1 Study subjects, sites and duration 2.1.1Study subjects People residing in Duc Hoa district, Long An province meet the selection criteria for the sample Patients infected with gastrointestinal strongyloidiasis Strongyloides larvae were collected from a patient's stool sample test Ivermectin pills single dose 2.1.2 Study sites (location) In the field works: communes Duc Lap Thuong, My Hanh Nam, Hiep Hoa, An Ninh Tay and Duc Hoa town of Duc Hoa district, Long An province In each commune or town, the location is people's residence and health station - Laboratory of medical parasitology, Pham Ngoc Thach university of medicine, Laboratory of Hematology and Immunization, Hospital for Tropical Diseases in Ho Chi Minh city - Genome sequencing at First BASE Laboratories-Axil Scientific, Malaysia 2.1.3 Study duration The study was conducted from July 2017 to November 2018 2.2 Study methods 2.2.1 Study design Cross-sectional descriptive study: describe infection prevalence and analyzing related factors Laboratory experiments Cross-sectional descriptive study describe case series and non-controlled treatment interventions 2.2.2 Research methods 2.2.2.1 Objective 1: Determine the actual situation and factors related to human Strongyloides spp infection in Duc Hoa district, Long An province in 2017-2018 Research content Determining the prevalence of intestinal strongyloidiasis in each commune / town of Duc Hoa district, Long An province Description and analysis: A number of demographic factors related to the prevalence of Strongyloides spp infection in people such as sex, age group, economic status and education level Some of the related behaviors such as agriculture job, toilet use and contact soil directly in daily life Techniques used in the study Interview technique of data collection Technique for diagnosis of Strongyloides spp infection  Modified Harada Mori culture (Sasa, 1986)  Stool direct smear technique 2.2.2.2 Objective 2: Determine component species of Strongyloides in human strongyloidiasis disease by morphological characterization and molecular biology Research content Describe the detection ability of direct stool test and modified Harada Mori culture technique in the first times Describe morphological characteristics of development stages of Strongyloides spp that collect from human feces Determine component species of Strongyloides in human strongyloidiasis disease in Duc Hoa district by a multiplex real-time PCR and gene sequencing Techniques used in the study Diagnostic technique: size measurement of morphological structures of Strongyloides spp larvae stage 1,2 or adults Multiplex real-time PCR technique, steps nested - PCR and gene sequencing 2.2.2.3 Objective 3: Describe the clinical symptoms, paraclinical and evaluate results of treatment for Strongyloides spp by ivermectin single dose Research content Describe the presence rate of clinical symptoms in patients who were diagnosed gastrointestinal strongyloidiasis before and after treatment: intermittent loosing diarrhea, abdominal pain, urticaria, weight loss, headache, cutaneous larvae migrans Describe and analyze the para-clinical parameters of patients who were diagnosed gastrointestinal strongyloidiasis: the number, ratio of eosinophils and serum ELISA diagnosis Determining the clinical and para-clinical therapeutic effect of single dose ivermectin and the presence rate of side effects Techniques used in the study Blood test technique to determine eosinophils ELISA test for detect specific antibody (IgG) against Strongyloides spp Stool test technique to evaluate treatment results: Apply a combination of direct smear technique and stool culture 2.2.3 Data processing Analyze the relation among the variables using 2 test, Fisher exact test, t test, OR with p 0.05 Economic status Yes < 0.01 2.08 Farmer Yes < 0.05 2.07 Using toilets (Unhygienic) Yes 3.30 < 0.01 Living habits (contact with soil) Yes < 0.05 2.69 Strongyloides spp infection in Duc Hoa district was associated with: male, over 60 of age, poor and nearby poverty economic status, farmer, using unhygienic toilets and contacted soil habits in daily life 3.2 Determine component species of Strongyloides in human strongyloidiasis disease 3.2.1 Survey pathogens by morphology Table 3.10 Analysis stool tests in human strongyloidiasis (n = 79) Percentage (%) Name of technique Number Direct smear 46 58.2 Modified Harada Mori culture 74 93.7 Coordinate both techniques 79 100 The direct smear test alone was only able to detect 58.2% of total cases, much lower than the culture technique Table 3.11: Morphology index of larvae stage (n = 79) Structure Mean ± SD Min – max Body length (µm) 279,9 ± 17,5 240.6 – 320.3 Horizontal size (µm) 18.47 ± 0.61 16.5 – 20.0 Length of esophagus (µm) 75.7 ± 5.1 64 – 90.1 Bucal cavity length (µm) 4.4 ± 0.3 3.9 – 5.3 Ratio esophagus length/body length (%) 27.1 ± 2.1 21.0 – 34.0 Pointed tail shape 79/79 (100%) 1st stage larvae: 100% with pointed tail, average length 279m, esophageal length averaged 27.1% compared to body length Table 3.12: Morphology index of larvae stage (n = 79) Structure Mean ± SD Min – max Body length (µm) 576.4 ± 24.9 510.0 – 632.0 Horizontal size (µm) 16.9 ± 1.1 15.3 – 19.6 Length of esophagus (µm) 244.7 ± 17.9 210.3 – 132.0 Bucal cavity length (µm) 4.5 ± 0.5 4.0 – 6.0 10 Ratio esophagus length/ body length (%) 42.5 ± 3.8 36.0 – 53.0 Horizontal size at endpoint of tail (µm) 2.6 ± 0.2 2.2 – 3.4 Endpoint of tail (blunt pointed/split 2) 11/68 (13.9 %/ 86.1 %) When cultured at day 3, 2nd larvae stage has slender shape, the endpoint of tail has blunt pointed or split in shaped Table 3.13: Morphology index of free-living male (n = 5) Structure Mean ± SD Min – max Body length (µm) 778.8 ± 27.7 740.8 – 812.6 Horizontal size (µm) 45.1 ± 1.7 43.4 – 47.6 Length of esophagus (µm) 131.3 ± 6.9 120.0 – 136.2 Bucal cavity length (µm) 7.1 ± 0.6 6.6 – 8.1 Ratio esophagus length/ body length (%) 17.0 ± 1.0 16.0 – 18.0 Length of genital spines (µm) 33.4 ± 0.9 32.1 – 34.4 Pointed tail shape (100%) Free-living male of Strongyloides spp had 778.8 µm average length, pointed tail Table 3.14: Morphology index of free-living female (n = 3) Structure Mean ± SD Min – max Body length (µm) 916.7 ± 21.6 892.6 – 934.2 Horizontal size (µm) 46.2 ± 1.7 44.2 – 47.5 Length of esophagus (µm) 130.6 ± 4.6 127.4 – 135.9 Bucal cavity length (µm) 6.8 ± 0.4 6.5 – 7.2 Ratio esophagus length/body length (%) 14.3 ± 1.2 14.0 – 15.0 Distance between vulva with head (% of 49 ± 1.0 48.0 – 50.0 body length) Free-living female of Strongyloides spp had 916.7µm average length, vulva was located near the middle of the body, slightly forward from to 1% of the body length 3.2.2 Results of real-time PCR in identification of Strongyloides spp In 79 samples of 2nd stage larvae were collected from 79 patients who infected with Strongyloides spp in Duc Hoa district DNA extraction was conducted according to the manufacturer's procedure, but only 70/79 samples response the requirements (88.6%) A total of 70 samples were included in the real-time PCR test Perform real-time PCR DNA Strongyloides spp on collected samples to identify genus of Strongyloides based on 28S rRNA gene sequences U3949 The identification of species S stercoralis based on Stro 18S gene sequences AF279916 and identified species S ratti based on the sequence Srat 28S gene location DQ14570 Table 3.15 Components of Strongyloides spp determined by real-time PCR (n = 70) 11 Species No Percentage (%) 94,2 S stercoralis 66 S ratti 2,9 Co-infection S stercoralis, S ratti 2,9 70 100 Total The ratio of S stercoralis was 97.1% (68/70) dominantly, of which 2.9% was co-infected with S ratti 3.2.3 Results of Nested - PCR and genetic sequencing 1002 bp 975 bp 500 bp 500 bp B A Figure 3.1 Electrophoresis products of PCR I (A) and PCR II on agarose gel 1,5%; M: scale of DNA 100 bp; C: Negative control (H2O); S: DNA sample of Strongyloides spp All 14 products of 2-step nested PCR included samples with S ratti presence and 10 S stercoralis random samples (obtained from real-time PCR), were sequenced genome Table 3.16 Analyzing results of sequence of 14 larvae samples in the study No 11 15 20 Highest similarity (%) 99,5 98,6 99,4 99,7 95,6 25 98,5/98 Code Gene code Species AB923888.1 AB923888.1 AB923888.1 AB923888.1 MK369923.1 AB923888.1/ AB453329.1 S stercoralis S stercoralis S stercoralis S stercoralis S stercoralis S stercoralis/ S ratti 12 10 11 12 13 26 35 42 47 50 54 91,3 100,0 100,0 99,2 100,0 98,0 LL999104.1 S stercoralis LL999088.1 S stercoralis LL999110.1 S stercoralis AB923888.1 S stercoralis MK369923.1 S stercoralis AB923889.1 S ratti AB923888.1/ S stercoralis/ S 65 99,3/98,0 AB453329.1 ratti 14 66 98,0 LN609412.1 S ratti The species components were similarity very high to the isolates that published in the gene bank Figure 3.2 Phylogenetic tree was built on group 10 S stercoralis larvaes Figure 3.3 Phylogenetic tree was built on group S stercoralis larvaes 3.3 Describe the clinical symptoms, paraclinical and evaluate results of treatment for strongyloidiasis by ivermectin single dose The total number of patients tested positive with Strongyloides spp was 79 cases Average age: 52.97 ± 27.64 (min - max = 22 - 84) 3.3.1 Clinical and paraclinical symptoms 13 The number of patients infected with Strongyloides spp completely without clinical symptoms was 10.1% Table 3.17 Clinical symptoms in human strongyloidiasis (n = 79) Symptoms No Percentage Detail No./Percentage (%) (%) Abdominal 58 73.4 Epigastric 33/79 (41.7%) pain A round the 17/79 (21.5%) navel Hypogastrium 8/79 (10.1%) Diarrhea, 33 41.8 Urticaria 45 57.0 The arms 36/79 (45.6%) Body 9/79 (11.4%) Headache 49 62.0 Lose-weight 11.4 CLM 3.8 Gastrointestinal symptoms had a high rate include: abdominal pain accounted 73.4% and diarrhea symptom was 41.8% Table 3.18 Percentage of patients with hyper-eosinophilia (n = 79) Value Number Percentage (%) Normal (< 500) 32 40.5 Eosinophil/µl Increase (≥ 500) 47 59.5 blood Total 79 100 (E) Mean = 694.56 ± 461.92 t test= 3.744; p value < 0.01; Distance of mean = 194.5; CT 95% (91.1 –298.0) Level of Normal (1500) 7.6 Total 79 100 There was 59.5% of patients had hyper- eosinophil in their blood The mean of eosinophil was 694.56, significantly different from the normal threshold p